This application claims priority to British Application No. 9716790.2, filed Aug. 7, 1997, the content of which is incorporated herein by reference.
The present invention relates to the production of insulin C-peptide from recombinant DNA molecules comprising multimeric copies of a gene sequence encoding said insulin C-peptide.
Insulin is a protein hormone involved in the regulation of blood sugar levels. Insulin is produced in the liver as: its precursor proinsulin, consisting of the B and A chains of insulin linked together via a connecting C-peptide (hereinafter this C-peptide derived from the proinsulin molecule is referred to as xe2x80x9cinsulin C-peptidexe2x80x9d). Insulin itself is comprised of only the B and A chains. Several recent studies indicate that the C-peptide has a clinical relevance (Johansson et al., Diabetologia (1992) 35, 121-128 and J. Clin. Endocrinol. Metab. (1993) 77, 976-981). In patients with type 1 diabetes, who lack endogenous C-peptide, administration of the peptide improves renal function, stimulates barrier function (Johansson et al., 1992 and 1993 supra).
Although not yet widely recognised, there is a growing awareness in the medical field,of a therapeutic utility for the insulin C-peptide. Accordingly, there is a need for a method for the ready synthesis of insulin C-peptides, economically and efficiently. Whilst methods for the chemical synthesis of peptides, e.g. by stepwise addition of amino acids on a solid support, are now well developed, they remain, despite automation, time-consuming and, more significantly, costly to perform, and may also be limited in terms of the maximum peptide length economically and reliably synthesisable. As an alternative, methods for peptide production by expression of recombinant DNA have been developed, although these too are not without their drawbacks e.g. in terms of yield.
Current production schemes for insulin C-peptide are based on the processing of proinsulin1 the precursor molecule for insulin and C-peptide, normally by the use of trypsin and carboxypeptidase B (Nilsson et al., (1996), J. Biotechnol. 48, 241-250); Jonasson et al., (1996) Eur. J. Biochem. 236, 656-661). Proinsulin was produced as a fusion protein that was capable of expression at high levels in E. coli, and the fusion protein was engineered in such a way that the fusion partner could be cleaved off simultaneously with the processing of proinsulin to insulin and C-peptide. Proinsulin was produced as a fusion protein with ZZ, a synthetic affinity fusion tag derived from staphylococcal protein A which binds IgG (Immuno-globulin) (Nilsson et al., (1987) Prot. Eng. 1, 107-113). This fusion tag was selected due to its stability to proteolysis, its IgG-binding capacity, its high expression levels and solubilizing properties. The chosen production strategy allowed the use of an affinity tag for efficient purification, after solubilization of inclusion bodies and subsequent renaturation, without the inclusion of additional unit operations for cleavage and removal of the ZZ affinity tag. The tag was demonstrated to be simultaneously cleaved off with the trypsin/carboxypeptidase B digestion of proinsulin to insulin and C-peptide. However, production of small peptides via the expression of large fusion proteins generally gives rather low yields, as the final product constitutes only a small part of the expressed gene product.
Shen in Proc. Natl. Acad. Sci. USA, 81, 4627-4631, 1984 describes a method for preparing human proinsulin by expression of a fused or unfused gene product comprising multiple tandemly linked copies of the proinsulin polypeptide domain. This gene product can be cleaved into single proinsulin units by cyanogen bromide treatment. It is proposed that human insulin can be prepared by cleavage of the proinsulin units with trypsin/carboxypeptidase. However, the problem of improving the yield of insulin C-peptide is not addressed.
There remains, therefore, a need for a recombinant expression method which improves the yield of insulin C-peptide, as an unfused product. The present invention addresses this need.
The present invention seeks to improve on existing methods for recombinant expression of peptides and essentially is based on the concept of increasing the amount of expressed target peptide (in this case an Insulin C-peptide) by expressing, as a single gene product, a multimer (i.e. a multimeric polypeptide) having multiple copies of the target peptide (insulin C-peptide), and then cleaving such a multimeric gene product (i.e. the multimeric polypeptide) to release the target peptide as individual monomer units.
In one aspect, the present invention thus provides a method of producing an insulin C-peptide, which comprises expressing in a host cell a multimeric polypeptide comprising multiple copies of a said insulin C-peptide, and cleaving said expressed polypeptide to release single copies of the insulin C,-peptide (i.e. to release the insulin C-peptide monomers from the multimer).
The multimeric polypeptide (gene product) is encoded by a genetic construct (in other words a nucleic acid molecule) comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide. The multiple copies, or repeats, are linked in the construct in such a manner that they are transcribed and translated together into a single, multimeric gene product (i.e. a multimeric polypeptide) i.e. in xe2x80x9cread-through formatxe2x80x9d e.g. the multiple nucleotide sequences are linked in matching reading frame in the construct. In essence, the genetic construct (nucleic acid molecule) advantageously comprises a concatemer of the insulin C-peptide encoding nucleotide sequence. Preferably, the genetic construct comprises tandem copies of the encoding nucleotide sequence. Such a genetic construct is thus prepared and is then introduced into a host cell in a standard manner, and expressed. The expressed gene product (polypeptide) may then be recovered and cleaved to release the insulin C-peptide monomers.
In a further aspect the invention thus provides a method for producing an insulin C-peptide, which comprises culturing a host cell containing a nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding a said insulin C-peptide, under conditions whereby the multimeric polypeptide of said nucleic acid molecule is expressed, and cleaving said expressed polypeptide to release single copies of said insulin C-peptide.
As used herein the term xe2x80x9cmultiplexe2x80x9d or xe2x80x9cmultimericxe2x80x9d refers to two or more copies of an insulin C-peptide or the nucleotide sequence which encodes it, preferably 2 to 50, 2 to 30 or 2 to 20, more preferably 2 to 15, or 2 to 10. Further exemplary ranges also include 3 to 20, 3 to 15 or 3 to 10.
Conveniently, the construct comprises 3 or more copies e.g. 3 to 7, or 5 to 7, copies of the nucleotide sequence encoding a insulin C-peptide. Ranges of 7 or more, for example 7 to 30, 7 to 20 or 7 to 15 may also be useful.
The term xe2x80x9cinsulin C-peptidexe2x80x9d as used herein includes all forms of insulin C-peptide, including native or synthetic peptides. Such insulin C-peptides may be human peptides, or may be from other animal species and genera, preferably mammals. Thus variants and modifications of native insulin C-peptide are included as long as they retain insulin C-peptide activity. The insulin C-peptides may be expressed in their native form, i.e. as different allelic variants as they appear in nature in different species or due to geographical variation etc., or as functionally equivalent variants or derivatives thereof, which may differ in their amino acid sequence, for example by truncation (e.g. from the N- or C-terminus or both) or other amino acid deletions, additions or substitutions. It is known in the art to modify the sequences of proteins or peptides, whilst retaining their useful activity and this may be achieved using techniques which are standard in the art and widely described in the literature e.g. random or site-directed mutagenesis, cleavage and ligation of nucleic acids etc. Thus, functionally equivalent variants or derivatives of native insulin C-peptide sequences may readily be prepared according to techniques well known in the art, and include peptide sequences having a functional, e.g. a biological, activity of a native insulin C-peptide. Thus, in terms of such activities, for example, insulin C-peptide is known to have an activity in stimulating Na+K+ATPase, which may underlie various of the therapeutic activities reported for C-peptide, e.g. in the treatment or diabetes or in the treatment or prevention of diabetic complications such as diabetic neuropathy, nephropathy and retinopathy. Fragments of native or synthetic insulin C-peptide sequences may also have the desirable functional properties of the peptide from which they derive and are hence also included. Mention may be made in particular of the insulin C-peptide fragments described by Wahren et al., in WO98/13384. All such analogues, variants, derivatives or fragments of insulin C-peptide are especially included in the scope of this invention, and are subsumed under the term xe2x80x9can insulin C-peptidexe2x80x9d.
Conveniently, the native human insulin C-peptide may be used and is shown in FIG. 2C (SEQ ID. NO. 1.)
In a further preferred embodiment of the method according to the invention, the gene construct will additionally comprise a sequence which encodes a fusion partner (fusion tag) e.g. which is capable of binding to matrices used during processing of the product of gene expression.
The term xe2x80x9cfusion partnerxe2x80x9d refers to any protein or peptide molecule or derivative or fragment thereof which is translated contiguously with the insulin C-peptide whose properties can be utilised in the further processing of the expressed fusion product.
The interaction between the fusion partner and the matrix may be based on affinity, chelating peptides, hydrophobic or charged interactions or any other mechanism known in the art. Conveniently, the fusion partner is one of a pair of affinity binding partners or ligands e.g. a protein, polypeptide or peptide sequence capable of selectively or specifically binding to or reacting with a ligand. Suitable fusion partners include for example streptococcal protein G and staphylococcal protein A and derivatives thereof, xcex2-galactosidase, glutathione-S-transferase and avidin or streptavidin, or a fragment or derivative of any aforesaid protein, which have strong affinities with immunoglobulin G. substrate analogues or antibodies and biotin respectively. Such interactions can be utilised to purify the fused protein product from a complex mixture. The ZZ fragment of protein A (see Nilsson et al., supra) is an example of a protein fragment which may be used. Histidine peptides can be used as fusion partners as they bind to metal ions e.g. Zn2+, Cu2+ or Ni2+ and elution may be performed by lowering the pH or with EDTA (Ljungquist et al. (1989) Eur. J. Biochem. 186, 563-569). Particularly preferred polypeptide fusion partners are a 25 kDa serum albumin binding region (BB) derived from streptococcal protein G (SpG) (Nygren et al. (1988) J. Mol. Recogn. 1 69-74) or other SpG-derived albumin binding tags (Stahl and Nygren (1997) Path. Bio. 45, 66-76). xc3x96berg et al., describe an expression vector, pTrp BB, (SEQ ID NO. 14) suitable for insertion of gene fragments for expression of a desired product as a fusion protein with BB (Proceedings of the 6th European Congress on Biotechnology, 1994, 179-182). These fusion partners have a strong affinity to albumin and therefore purification of the expressed fusion protein can be based on ligand affinity chromatography e.g. using a column charged with albumin. The albumin is preferably immobilised on a solid support.
Any convenient means may be used to achieve the cleavage step, i.e. the cleavage of the monomeric insulin C-peptides from the multimeric polypeptide i.e. from the expressed gene product, and optionally from the fusion partner if present. Conveniently, this may be achieved using enzymes. Preferably, the initial product of gene expression, i.e. the multimeric polypeptide or the fusion product or fusion protein, which comprises the fusion partner and multiple copies (monomers) of the insulin C-peptide, is cleaved by one or more proteolytic enzymes in a single process step to yield unfused single copies of the insulin C-peptide. A combined treatment with trypsin and carboxypeptidase B (e.g. from bovine, porcine or other sources) is a particularly preferred method of obtaining the desired cleavage products. Trypsin cleaves the proteins C-terminally of each arginine residue and carboxypeptidase B removes the C-terminal arginine present on each peptide after trypsin digestion. Conditions for achieving proteolytic cleavage are well known in the art, as are a range of other suitable proteolytic enzymes such as Subtilisin (including mutants thereof), Enterokinase, Factor Xa, Thrombin, IgA protease, Protease 3C, and Inteins. It has been found, for example, that incubation of the expressed gene product with the proteolytic enzymes (e.g. trypsin and carboxypeptidase B) for 60 minutes is sufficient for complete processing of the expressed protein. Conveniently, 5 minutes incubation time may be sufficient for adequate processing of the fusion protein such that no fusion or multimeric protein is detectable by conventional SDS PAGE. Alternatively, the initial product of gene expression may be cleaved by chemical reagents such as CNBr, hydroxylamine or formic acid.
Depending on the precise nature of the insulin C-peptide and nucleic acid molecule (genetic construct) used, the cleavage sites e.g. for proteolysis may be present naturally, or they may be introduced by appropriate manipulation of the genetic construct using known techniques e.g. site-directed mutagenesis, ligation of appropriate cleavage site-encoding nucleotide sequences etc.
Conveniently, the multimeric expressed polypeptide may include a linker region i.e. a linker residue or peptide incorporating or providing a cleavage site. Advantageously, the cleavage site comprises a cleavable motif recognised and cleaved by a proteolytic enzyme. Linker regions may be incorporated between each xe2x80x9cmonomerxe2x80x9d peptide in the multimeric construct, and/or optionally also between the fusion partner if present and a monomer peptide. Advantageously, each monomer peptide may be tandemly arranged with a linker region. Advantageously, the insulin C-peptide monomers in the multimer are flanked by appropriate linker sequences to ensure cleavage and release of insulin C-peptide free of any linker region residues. The linker region may comprise from 1 to 15 e.g. 1 to 12 or 1 to 10 amino residues, although the length is not critical and may be selected for convenience or according to choice. Linker regions of from 1 to 8, e.g. 1, 5 and 7 may be convenient. The individual linker region within each construct may be the same or different, although for convenience they are generally the same. Thus, for example, for cleavage by the combination of trypsin and carboxypeptidase B, linkers beginning or terminating in arginine residues may be provided.
An alternative linker may comprise the amino acid lysine, either solely or as part of a longer sequence and may also be cleaved by the trypsin/carboxypeptidase B combination.
For inclusion between insulin C-peptide monomers, such linkers may advantageously start with and terminate in such a cleavage site e.g. an arginine residue at both their N and C termini, to ensure release of an insulin, C-peptide monomer without any additional amino acids. For inclusion between the fusion partner and/or at the end of the insulin C-peptide multimer, a single cleavage site (e.g. Arg) may be present at the appropriate terminus of the linker, (or correspondingly at an appropriate site for cleavage, depending on the precise linker sequence and cleavage enzymes used).
Exemplary representative linker regions include -RTASQAR- (SEQ ID NO. 2) for inclusion between C-peptide monomers, -ASQAR- (SEQ ID NO. 3) between the fusion partner and a C-peptide multimer and -RTASQAVD (SEQ ID NO. 4) at the end of the multimer.
As mentioned above, standard methods well-known in the art may be used for the introduction of linker sequences.
A further aspect of the present invention is a nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide, wherein said nucleic acid molecule encodes a multimeric polypeptide capable of being cleaved to yield single copies of said insulin C-peptide.
Alternatively viewed, this aspect of the invention can be seen to provide a nucleic acid molecule comprising a concatemer of a nucleotide sequence encoding an insulin C-peptide.
The various aspects of the invention set out above (and below) include embodiments where the multimeric polypeptide (gene product) does not include both an insulin A and an insulin B peptide, or where the nucleic acid molecule does not encode both an insulin A and B peptide. More particularly, in such embodiments, where the number of copies of insulin C-peptide in the multimeric polypeptide, or encoded by the nucleic acid molecule, is two, the multimeric polypeptide does not include, or the nucleic acid molecule does not encode, both insulin A and B peptides.
In a particularly preferred embodiment of the invention, the nucleic acid molecule will additionally comprise a nucleotide sequence which encodes a fusion partner which assists in the further processing of the encoded multimeric polypeptide e.g. which is useful for purification of the expressed protein product. The gene encoding the fusion partner will be in the correct position and orientation to be translated together with the multiple copies of the insulin C-peptide to form, initially, a single fused peptide. Suitable fusion proteins are discussed above.
Advantageously, the nucleic acid molecule will also comprise one or more nucleotide sequences encoding linker regions comprising cleavage sites, as discussed above.
As exemplary of nucleic acid molecules according to the invention may thus be mentioned those encoding a polypeptide of Formula I)
H2N-A-(Cxe2x80x94X)nxe2x80x94COOHxe2x80x83xe2x80x83(I)
wherein
C is an insulin C-peptide;
A is a bond, or a group F, wherein F is a fusion partner, or a group xe2x80x94(Fxe2x80x94X)xe2x80x94;
X is a linker region comprising at least one cleavage site, each X being the same or different; and
n is an integer of 2 to 50.
This aspect of the invention includes an embodiment wherein Formula (I) includes the proviso that when n=2, said polypeptide (I) does not comprise an insulin A and B chain.
Insulin C-peptides (group C), fusion partners (group F) and linker regions (group X) may be as defined above. Likewise n may be as defined above in relation to the terms xe2x80x9cmultiplexe2x80x9d and xe2x80x9cmultimericxe2x80x9d.
The nucleic acid molecule or genetic construct useful in the methods of the invention will preferably contain a suitable regulatory sequence which will control expression in the host cell. Such regulatory or expression control sequences include, for example, transcriptional (e.g. promoter-operator regions, ribosomal binding sites, termination stop sequences, enhancer elements etc.) and translational (e.g. start and stop codons) control elements, linked in matching reading frame to the coding sequences.
Any suitable host cell may be used, including prokaryotic and eukaryotic cells and may be selected according to the chosen expression system e.g. bacterial, yeast, insect (e.g. baculovirus-based) or mammalian expression systems. Very many different expression systems are known in the art and widely described in the literature. For example, E. coli can be used as host cells for peptide production, in which case, the regulating sequence may comprise, for example, the E. coli trp promoter. Other suitable hosts include Gram-negative bacteria other than E. coli, Gram-positive bacteria, yeast insect, plant or animal cells e.g. genetically engineered cell-lines.
Expression vectors which comprise the nucleic acid molecules described above constitute a further aspect of the present invention.
Any convenient vector may be used to achieve expression according to the methods of the invention and very many are known in the art and described in the literature. Suitable vectors thus include plasmids, cosmids or virus-based vectors. These vectors, which are introduced into the host cells for expression, are however, preferably plasmid, phage or virus vectors. The vectors may include appropriate control sequences linked in matching reading frame with the nucleic acid molecules of the invention. Other genetic elements e.g. replicons, or sequences assisting or facilitating transfer of the vector into the host cell, stabilising functions, e.g. to assist in maintenance of the vector in the host cell, cloning sites, restriction endonuclease cleavage sites or marker-encoding sequences may be included according to techniques well known in the art. The vectors may remain as discrete entities in the host cell or may, in the case of plasmid insertion vectors or other insertional vectors, be inserted into the host cell chromosome. Random non-specific integration into the host chromosome is possible, although specific homologous integration is preferred. Techniques for this are known in the art (see e.g. Pozzi et al. (1992) J. Res. Microbiol. 143, 449-457 and (1996); Gene 169, 85-90). The integration is xe2x80x9chomologousxe2x80x9d because the plasmid insertion vector comprises a segment of host cell chromosomal DNA.
Representative exemplary plasmids suitable for expressing genetic constructs, or nucleic acid molecules according to the invention include pTrpBB (xc3x96berg et al., supra) or derivatives thereof. Alternatively such plasmids may be modified to remove sequences encoding the fusion partner if desired. Any high-copy number vector incorporating a Trp-promoter or similar may be used.
A variety of techniques are well known in the art and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression e.g. bacterial transformation techniques, transfection, electroporation. Transformed or transfected eukaryotic or prokaryotic host cells i.e. host cells containing a nucleic acid molecule according to the invention and as defined above, form a further aspect of the invention.
As described in more detail in the Examples, expression vectors, specifically plasmids, harbouring the nucleic acid molecules of the invention have the advantage of genetic stability in their hosts; no genetic instability was detected in plasmids prepared from cultures grown to high cell densities, as assessed by restriction mapping.
A further aspect of the present invention provides a method for the production of a nucleic acid molecule which encodes a multimeric polypeptide comprising multiple copies of an insulin C-peptide, wherein the expressed multimeric polypeptide is capable of being subsequently cleaved to yield single copies of the insulin C-peptide, said method comprising generating a nucleic acid molecule comprising multiple copies of a nucleotide sequence encoding an insulin C-peptide, linked in matching reading frame.
There are a number of techniques known in the art for generating multimeric copies of a gene or gene fragment which can be used in the methods of the present invention. For example, synthetic DNA fragments can be head-to-tail polymerised utilising designed single-stranded non-palindromic protruding ends. The polymerised DNA fragments can then be directly ligated to matching protrusions resulting from enzymatic restriction (Ljungquist et al. (1989) Eur. J. Biochem. 186, 563-569). Other methods to achieve multimerisation of gene fragments are based on the use of class IIS restriction enzymes such as Bsp MI (Stxc3xa5hl et. al (1990) Gene 89, 87-193) or Bsm I (Haydn and Mandecki (1988) DNA 7, 571-577). Alternative strategies involve polymerisation of the gene construct and ligation of adapter molecules containing restriction sites to allow further subcloning (xc3x85slund et al. (1987) Proc. Natl. Acad. Sci. USA 84, 1399-1403 and Irving et al. (1988) in Technological Advances in Vaccine Development, A. R. Liss Inc., New York 97-105). Methods for de novo synthesis of genes are also known, involving the use of the polymerase chain reaction (PCR), that would be suitable for the generation of multimeric gene fragments (Majumder (1992) Gene 110, 89-94) and Nguyen et al. (1994) in Advances in Biomagnetic Separation, Eaton Publishing Co., Natick 73-78).
In a preferred embodiment of the method according to the invention, the purified gene fragments (i.e. nucleotide sequences encoding an insulin C-peptide) are allowed to polymerize in a head-to-tail fashion (multimerise), due to designed non-palindromic protrusions and are then ligated into a plasmid digested by a restriction enzyme, preferably Sfi I.
In a particularly preferred embodiment, a plasmid comprising a nucleotide sequence (e.g. a gene fragment) encoding an insulin C-peptide is digested to excise the said sequence origene fragment and after multi-merisation of the sequences or gene fragments they are ligated back into the digested plasmid. Transformants may advantageously be screened using a PCR-screening technique (Stxc3xa5hl et al. (1993) Biotechniques 14, 424-434) which amplifies the segment encoding one or more copies of the insulin C-peptide. The PCR amplified fragments can be compared by agarose gel electrophoresis. In a further preferred embodiment, gene fragments encoding a desired number of concatamerized insulin C-peptides e.g. three or seven, are isolated and ligated into a further plasmid which has been digested using the same restriction enzyme as was used to excise the fragment encoding the insulin C-peptide. Most preferably, this later plasmid, which will be used for transformation of host cells, additionally comprises a suitable promoter and a sequence encoding a suitable fusion partner for the insulin C-peptide.
Further aspects of the invention include the products of the aforementioned methods, namely an insulin C-peptide multimer and the individual C-peptides released from said multimer by cleavage.
In particular, this aspect of the invention provides a multimeric polypeptide comprising multiple copies of an insulin C-peptide cleavable to release single copies of said insulin C-peptide. Optionally, the multimeric polypeptide may additionally comprise a fusion partner, and/or linker regions comprising a cleavage site flanking each said C-peptide monomer.
Also provided is a method for producing a multimeric polypeptide comprising multiple copies of an insulin C-peptide cleavable to release single copies of said insulin C-peptide, said method comprising culturing a host cell containing a nucleic acid molecule encoding said multimeric polypeptide under conditions whereby said multimeric polypeptide is expressed, and recovering the expressed multimeric polypeptide.
The host cells may be cultured using techniques known in the art e.g. batch or continuous culture formats.
The multimeric gene product. or polypeptide may be recovered from the host cell culture using standard techniques well known in the art, e.g. standard cell lysis, and protein purification techniques. As mentioned above, where a fusion partner is included in the multimeric polypeptide, purification may readily be achieved based on affinity binding of the fusion partner.
A variety of techniques are known in the art for isolating proteins or polypeptides from cells or cell culture medium, both native and recombinantly expressed, and any of these may be used. Cell lysis to release intracellular proteins/polypeptides may be performed using any of the many methods known in the art and described in the literature, and if necessary further purification steps may be performed, again based on techniques known in the art, depending on whether batch or continuous culture methods are used.
Heat treatment methods for the lysis of cells and recovery of polypeptides have been found to be particularly effective in the case of the insulin C-peptide multimeric polypeptides of the present invention, for example the method described in WO90/00200 and modifications thereof. Such methods involve heating the host cell-containing culture medium e.g. for 50-100xc2x0 C. for a period of time, generally not exceeding 1 hour, whereby the expressed polypeptide is released into the medium, advantageously in substantially pure form. This is believed to result from a selective release of the expressed polypeptide. In particular, it has surprisingly been observed that such a method works well in the case of soluble polypeptide products which are stable to the heat treatment, whether recombinant or not (and the method may thus be of more general applicability), but especially in the case of the insulin C-peptide multimeric polypeptide of the invention, where surprisingly high yields of high purity product may be obtained. Then, for example, such heat treatment may take place by heating at 80-100xc2x0 C. e.g. 85-99xc2x0 C. or 90-95xc2x0 C. for 5-20 minutes, e.g. 8-10 minutes, and cooling thereafter, e.g. to 0-4xc2x0 C. or on ice.
Following recovery of the multimeric polypeptide, it may be cleaved to release the individual insulin C-peptide monomers. Accordingly a further aspect of the invention provides a method for producing an insulin C-peptide, said method comprising cleaving a multimeric polypeptide as defined above, to release single copies of said insulin C-peptide.
Following cleavage of the multimeric polypeptide as discussed above to yield individual C-peptide monomers, these may also further be purified, e.g. to homogeneity (e.g. as demonstrated by SDS-PAGE) using well known standard techniques of purification e.g. ultrafiltration, size-exclusion chromatography, clarification, reversed-phase chromatography etc.
A further aspect of the present invention is the use in therapy of the cleaved peptide products of the methods described above. The cleaved insulin C-peptide can be used in the treatment of type 1 diabetes and/or diabetic complications. Also within the scope of the a present invention therefore, is a method of treating type 1 diabetes or the complications thereof comprising administration of insulin C-peptide prepared by any of the methods described above.